The established procedure for quantitative assay of proteases is based either on immunological techniques or reaction with some kind of substrate--biological or synthetic.
Immunological methods utilizing the antibody-antigen reaction can, of course, be used with all proteases with available specific antibodies. There are, however, two important drawbacks--the method is time-consuming because the result does not appear till after 1-24 hours, and it is not sufficiently discriminative in so far as the protein--functionally active or not--reacts with its antibody as long as the antigenic structure (not determinant) is intact. Thus, the immunologically assayable quantity of a protein is equal to or larger than the quantity of functionally active protein. The advantage of the method, however, is its specificity and high sensitivity.
Examples of reactions with natural substrates used for protein determination are: the reaction of thrombin with fibrinogen in coagulation, the plasmin reaction with fibrin in fibrinolysis, and the elastase reaction with elastine. Thus, the usual diagnostic methods used in the field of coagulation are mostly based on the fact that plasma samples are activated under standardized conditions and the final reaction step is the reaction of thrombin with fibrinogen. In this procedure, fibrinogen is transferred to insoluble fibrin, the forming of which is the final step of the assay. Another example is the horseshoe crab haemocytelysate which coagulates after endotoxin activation of its clotting enzyme (Limulus test for endotoxin).
Other methods for the determination of proteases use biological proteins which are not in themselves natural substrates for the protease in question. Examples: casein, haemoglobin, gelatine and insulin.
The disadvantage of working with protein substrates is the usually very limited sensitivity and lack of specificity of these methods. Further, they are difficult to standardize, are rather time-consuming and cannot easily be automatized.
Synthetic, low-molecular weight substrates based on amino acids or short peptides provided with a marker easily determinable and easily split by protease have, in recent years, considerably facilitated the methods for quantification of proteases by eliminating several of the above-mentioned disadvantages.
These substrates--being organic synthetic chemicals--have in common the quality of being easily standardized. Further, there is the possibility of making these substrates more or less specific for various types of proteases by a tactical choice of the amino acid or amino acid sequence linked to the marker. Thus, the choice of a suitable substrate structure can be adapted to the actual demand. In addition, these kinds of substrates permit the use of markers to be liberated in the protease reaction and, thus, easily quantified in an objective measuring equipment (spectrophotometer, fluorimeter, radioactivity counter).
Two types of derivatives have been utilized as substrates, esters or amide derivatives of the markers. As a rule, the ester substrates have proved to be more easily split than the corresponding amide derivatives. At the same time, however, splitting of amide derivatives is a physiologically and biochemically better model of the specific function of proteases which is splitting of amide bindings. In addition, the ability of a protease to split esters and amides is in certain cases related to different reaction centers within the molecule which means that, in such a case, amide substrates are required in order to be able to determine the physiological function.
Among the markers used in synthetic substrates of the amide type according to the foregoing, derivatives of p-nitroaniline (pNA) for photometric quantification and of .beta.-naphthylamin (.beta.NA) as well as of 2-amino-4-methylcoumarin (MCA) for fluorometric quantification are the most commonly used and described, according to information available from the literature and accessible patent documents.
Substrates with radioactive labelled markers are sporadically mentioned in the literature but, as the properties of an intact substrate and a split-off marker do not differ in regard to radioactivity, a separation step is required before the assay. This complication, in addition to the risk involved in the handling of radioactive material and the problem of lack of stability of the substrate due to the natural decompositions of the radioactive isotope introduced in the substrate, has made this kind of substrate useful only in very special instances.
Comprehensive studies have been carried out with synthetic substrates--usually provided with pNA or MCA markers--in order to establish structures which, with a limited number of amino acids in the peptide part (usually 2 to 4), provide an optimum of sensitivity and specificity for a number of endo- and exogenic proteases important from the clinical point of view. It has been found that the pNA- or MCA marker, respectively, is of no decisive significance to the general properties of the substrates. Thus, the specificity profile of a substrate, that is, its relative reactivity to various enzymes, is mainly governed by the peptide structure. Further, the differences in K.sub.m and k.sub.cat -values do not give differences in substrate turnover rate in terms of "moles substrate converted per time unit" larger than usually about 10 times. If, furthermore, the solubility of each substrate allows for substrate concentrations stronger than twice the K.sub.m -concentration, only k.sub.cat is essential. The differences mentioned in the literature may, in certain cases, also be explained by different buffer systems and contents of organic solvents.
Thus, the capacity of each substrate for assaying low levels or small amounts of a certain enzyme is primarily determined by the possibility to determine low levels or small volumes of each marker after its release from the substrate.
The smallest content of MCA in pure aqueous solutions which can be determined photometrically or fluorimetrically would be 10.sup.-6 M or 10.sup.-9 M respectively. But as also intact substrate in considerably higher concentrations is present in the solution in protease assays with chromogenic and fluorogenic synthetic substrates, the sensitivity of the methods is limited by the background effect supplied by unsplit substrate--in particular when minute concentrations of released marker are to be determined.
This difference is due to the fact that the properties of the marker are not the same when it is free and when it is acylated by an amino acid or a peptide. Whichever amino acid or peptide forms the acyl group is of minor imporance. Thus, the difference between an MCA substrate and free MCA is 500 to 700 that is the fluorimeter reading is the same for 1,5--2.multidot.10.sup.-7 M MCA as for a 1.multidot.10.sup.-4 M MCA substrate solution (Zimmermann et al., Anal Biochem 70, 258 (1976), ibid. 78, 47 (1977), Proc Natl Acad Sci USA 75, 750 (1978). The linear response is said to be obtained for a 500-fold concentration range.
The smallest determinable volume depends on the type of appliance used but usually a volume of 0.5 to 3 ml is required for these photo- and fluorometric methods--volumes of less than 0.5 ml require special equipment for exact determination because both types of marker require a certain amount of radiation to give an exact measure of the light quantity absorbed or emitted.